Decoding functional hematopoietic progenitor cells in the adult human lung

The bone marrow is the main site of blood cell production in adults, however, rare pools of hematopoietic stem and progenitor cells with self-renewal and differentiation potential have been found in extramedullary organs. The lung is primarily known for its role in gas exchange but has recently been described as a site of blood production in mice. Here, we show that functional hematopoietic precursors reside in the extravascular spaces of the human lung, at a frequency similar to the bone marrow, and are capable of proliferation and engraftment. The organ-specific gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune, megakaryocyte/platelet and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to a vascular-rich alveolar interstitium niche. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.


Introduction
Hematopoietic stem cells (HSCs) are self-renewing cells residing in the bone marrow (BM) that are ultimately responsible for production of all mature circulating blood cell lineages. 1Despite the burden of maintaining hematopoiesis, HSCs are a rare cell type in the BM accounting for <0.01% of nucleated cells. 2,3They occupy and are maintained by a speci c BM niche yet are able to exit the BM environment and enter the circulation, although the explanation of this behavior is largely unknown. 4HSCs are central to the pathogenesis of serious disorders including myelodysplasia, acute and chronic leukemia, aplastic anemia 5 and clonal hematopoiesis 6 , and HSC transplantation can be a life-saving therapy.Previously, we discovered extravascular hematopoietic stem and progenitor cells (HSPCs) in the adult mouse lung capable of engraftment in the BM and multi-lineage hematopoiesis. 7Motivated by this background, in this study we sought to determine if HSPCs occupied the adult human lung-an organ with a vast vasculature that contains a wide-ranging repertoire of stromal and immune cells.

The adult human lung contains functional HSPCs
We had the unique opportunity to receive matched adult human lungs, vertebral bodies (BM), and peripheral blood (PB) freshly recovered from deceased research donors (Table S1).The lungs were extensively perfused at the time of collection, and we selected healthy-appearing lung tissue from the upper lobes for experiments (Figure S1A).][10] Notably, our lineage panel contained markers for mature immune, endothelial, and epithelial cells, allowing for the phenotyping of rare Lin -CD34 + cells.We discovered a distinct population of multipotent progenitors (MP; (CD34 + /CD38 -) in the lung and BM and very few of these cells in the peripheral blood (PB) (Figure 1B).The lung and BM contained cells with surface staining consistent with HSCs (CD34 + /CD38 -/CD90 + /CD45A -) but these cells were absent in the PB.
Multipotent progenitor (MPP) cells (CD34 + /CD38 -/CD90 -/CD45A -) were observed in all three tissues.CMP (CD34 + /CD38 + /CD45A -/Flt-3 + ), GMP (CD34 + /CD38 + /CD45A + /Flt-3 + ), and MEP (CD34 + /CD38 + /CD45A -/Flt-3 -) cells were observed in all three tissues but were less common in the lung (Figure 1B).Overall, the BM and PB had nearly identical proportions of hematopoietic progenitors, while Lin -lung cells were enriched for immunophenotypic HSCs and MPPs (Figure 1C).To rule out residual blood as a source of HSPCs in perfused lungs, we estimated the numbers of progenitors in equivalent volumes of blood and lung tissue, yielding lung HSPC numbers that could not be explained by retention of intravascular blood in lungs (Figure S1B).Further, the distinct proportions of cell subsets in lung and blood indicates a tissue-speci c progenitor composition (Figure S1C).Remarkably, the frequency of the immunophenotypic HSC/MPP pool in the lung is similar to the BM (Figure 1D), while the pool of more committed hematopoietic progenitor cells is much smaller in the lung (Figure 1D, Figure S1D).We also tested for the effects of donor age and gender on our results and found that increasing age was associated with fewer numbers of HSPC pools in the BM but not in the lung (Figure S2A-B).Gender was not associated with changes in HSPC frequency in the BM or lung (Figure S2C-D).
Since broblasts are generally lineage-negative cells, but some can be CD34-positive 11 , we included a marker for broblasts (PDGFRα) in our lineage panel to determine if broblast contamination could affect our results, but we did not detect any changes in HSPC frequencies between our two lineage panels (Figure S3A-C).
We next tested the functional capacity of lung HSPCs using in vitro colony forming assays.We plated Lin -cells from the lung or BM in MethoCult TM and observed a variety of colonies from both tissues.The Lin -cells from the lung produced overall fewer colonies, but with a signi cant increase in the relative proportion of erythroid colonies (BFU-E) (Figure 1E).Following morphologic colony assessment, we used ow cytometry (Figure S4A-B) to con rm cellular colony composition and found that lung colonies were enriched with cells expressing the erythrocyte marker GlyA (Figure S4C).We also plated Lin -cells in MegaCult TM to test the potential to produce megakaryocytes, a cell population that we previously described as resident cells in the mouse lung. 7Both BM and lung cells were capable of producing megakaryocyte colonies although the lung produced fewer and small colonies (Figure 1F).The fewer colonies observed from lung cells may relate to overall reduced cell cycling compared to the BM (Figure 1G).The molecular cues that regulate the transition to activity in pulmonary HSPCs are not known and could be different from conditions in classical in vitro CFU assays.Together, we conclude that the human lung contains functional HSPCs that exhibit an erythroid bias.

Human lung-derived hematopoietic progenitors have engraftment potential
We next tested whether HSPCs isolated from the lung are capable of engraftment when xenotransplanted into immunode cient mice. 12We chose NSG-SGM3 mice (human SCF, GM-CSF, IL-3) to support human myeloid cell engraftment 13 for these experiments in which we adoptively transferred magnetic-enriched Lin -cells from the lung or BM into mice after sublethal irradiation 14 .Given that the production of erythroid cells in xenograft models is not reliable due to the lack of cross-reactivity between mouse EPO and the human EPO-receptor, mice received recombinant human erythropoietin injections in the nal three weeks of the experiment (Figure 2A) as described previously. 125][16] Engraftment was rigorously de ned as the presence of human CD45 + cells, using two different antibody clones, with the threshold for engraftment set to ≥0.01%CD45 ++ cells of all CD45 + cells (mouse and human) with at least 30 cells recorded in the CD45 ++ gate for BM and lung, and ≥ 15 cells for PB 12 given that low levels of engrafted human cells are expected from previous studies. 16amples of positive and negative engraftment (sublethal irradiation without adoptive cell transfer) are shown in Figure 2B.Overall, 6/7 mice with BM-derived HSPCs and 5/7 mice with lung-derived HSPCs engrafted in the BM and in the lung tissue (Figure 2C, Figure S5A-B).In the PB, engraftment was observed in 4/7 mice with BM-derived HSPCs and 2/7 mice with lung-derived HSPCs (Figure 2C).Examples of BMor lung-derived cells in the mouse BM or lung are shown in Figure 2D.We also determined erythroid engraftment using the gating strategy in Figure 2E to detect CD45 -, hGlyA + , hCD71 + cells and observed similar erythroid engraftment of BM-or lung-derived HSPCs in the mouse BM, lung, or PB (Figure 2F-G).Similar to BM-derived HSPCs, lung-derived HSPCs were capable of multilineage blood cell production (Figure 2H, Figure S5C-E).

Human lung HSPCs have unique transcriptional programming
The availability of matched lung and medullary HSPCs allowed us to directly compare their transcriptional pro les using single-cell RNA-sequencing (scRNA-seq, Figure S6).We integrated all samples using Harmony 17 and generated a batch-corrected UMAP to identify clusters of transcriptionally similar cells.By using the function ' ndConservedMarkers' we identi ed genes that were consistently expressed across BM and lung-derived cells and annotated clusters based on a reference data set. 18,19imensionality reduction yielded a visual representation consistent with HSC and MPP production of progenies with progressive commitment to more differentiated fates (Figure 3A, Figure S6A-B).To validate our annotation, we generated co-regulated modules of differentially expressed genes using Monocle3.20 The module highly speci c for HSCs contains genes such as AVP, SPINK2, SELL and HOPX and was strongly expressed in cells from both the BM and lung (Figure 3B, Figure S6C-D).We ordered the cells along a pseudotime trajectory to reconstruct their developmental path for each tissue individually, suggesting a relationship between HSCs and stromal cells in the lung that was absent in cells derived from the BM (Figure 3C).As previously discussed, some pulmonary broblasts are CD34 + and these results could point to the ontogeny of a subset of lung stromal cells.
Next, we compared the differential gene expression between medullary and lung cells within the HSC/MPP cluster and plotted the median expression of both cells on a scatter plot (Figure 3D).Using the Wilcoxon rank-sum test in Seurat's 'FindMarkers' we identi ed 50 genes that were upregulated in lung HSCs and 10 genes that were higher in BM cells (Figure 3D).2][23][24] We attributed the highest biological relevance to genes that were upregulated in most of the cells, as validated by analyzing the distribution of gene expression values (Figure 3F-H).HSCs from the lung have unique features (Figure 3F) and share characteristics of the hematopoietic lineage (Figure 3G), while as expected, cells from the BM have high expression of classical stem cell genes (Figure 3H). 18xt, we performed single-sample Gene Set Enrichment Analysis (ssGSEA) to identify pathways that are differentially regulated between cells from the lung and the BM.The enrichment scores were calculated across all individual cells and tissues for gene set collections from the Molecular Signature Database (H: hallmark, CP: canonical pathways, C5: ontology).In the gene sets analyzed, repeatedly pathways associated with erythropoietin (EPO) signaling, platelet function, and immune responses were found to be upregulated in pulmonary HSCs (Figure 3I).Side-by-side comparison of selected pathways indicates that HSCs from the lung are enriched for megakaryocyte (R-HSA-8936459) and EPO-induced erythroblast (R-HSA-9027277, R-HSA-9006335) differentiation, as indicated by higher normalized enrichment scores (NES) (Figure 3J).Our nding of increased EPO signaling pathways in lung HSCs is consistent with our data in Figure 1E on the erythroid-biasing of lung colonies.Our nding of platelet and megakaryocyteskewing of lung HSCs is provocative in light of our previous work on platelet production in the lung and tissue-resident immune-like megakaryocytes. 7,25Additionally, we found in ammatory signaling to be upregulated in pulmonary HSCs (Figure 3K), suggesting that these cells could impart unique immunological functions of their progeny.
A small cluster of cells has recently been suggested as hematopoetic stem cell population in an analysis of the healthy and diseased human lung tissue based on CD34, SPINK2, STMN and PRSS57 expression 26 , but the self-renewal and differentiation potential, and the location of these cells is not known.Also, CD34 + cells can be found in endothelial, lymphatic and broblast clusters (Figure S7A-B).To identify potential HSCs based on their transcriptomic pro le, we used UCell scoring to nd Lin -, CD34 + cells with an HSC signature in a dataset combining 9 human lung scRNA-seq studies (Human Lung Cell Atlas V2, HLCA) 27 (Figure S7C-D).We projected these cells on our UMAP structure of hematopoietic stem and progenitors cells from the lung and BM (Figure 3A) in Figure S7E and mapped 43 cells to the HSC/MPP cluster (Figure S7F) across all integrated datasets in the HLCA (Figure S7G).We conclude that given their rarity and the co-expression of CD34 in multiple lung cell types, lung HSCs are mostly masked and overlooked when using standard unsupervised clustering techniques.

HSPCs in the lung reside in the extravascular tissue
The lung is composed of diverse cell entities, such as epithelial, endothelial, stromal, and immune cell subpopulations that could provide a unique niche for HSPC maintenance and differentiation. 19To map HSPCs to their lung compartment, we used a spatial transcriptomics approach based on combinatorial single-molecule uorescent in situ hybridization (Figure 4A).We designed a marker panel that characterizes HSPCs as well as the common cell entities of the lung (Table S3).Following QuPath-based cell segmentation, we ltered for putative HSPCs de ned by CD34 + positivity, expression of HSPCassociated transcripts and negativity for marker genes of other lung cell entities (Figure 4B, Figure 8A, B).We visually validated all candidate cells and assigned them to their anatomic location.Over 90% of cells matching the criteria for HSPCs localized to the extravascular lung, with the majority of the cells in the alveolar interstitium or in proximity of bronchi (peribronchial) and vasculature (perivascular) (Figure 4C).
To de ne the neighborhood of HSPCs on the cellular level, we performed unsupervised clustering of the cells generated via segmentation using their transcript expression values (Figure 4D, Figure S8C and Figure S9A-D).Based on this annotation, we used Squidpy for co-occurrence analysis across all samples 28 suggesting that the immediate HSPC niche is mainly formed by endothelial cells, although epithelium and broblasts have a steady presence (Figure 4E-F).

Discussion
For many years, HSCs were viewed as unbiased cells that initiated hematopoietic development and specialization.Enabled by new technologies, our understanding of hematopoiesis has been re ned to include the possibility that developmental biases may be present even in these most undifferentiated cells, such as with megakaryocyte-biased HSCs. 9However, the mechanisms responsible for this early biasing or specialization are not clear. 29,30Here, we propose that the traditional view of HSC residency in the BM should be reconsidered to include extramedullary tissues, such as the lung.Indeed, and remarkably, we found an equal frequency of multipotent progenitors residing in the bone marrow and lung.Due to the low frequency, we used approaches to enrich for the presence of HSPCs in our studies, which were not done in previous studies and likely enabled the pro ling of this rare subset of cells amongst the >30 different lung cell types.
It is clear from our studies that the lung HSPCs have unique features compared to their medullary counterparts, but also to HSPCs found at other extramedullary sites (peripheral blood and spleen 31 ).Chief amongst these, and perhaps obvious from our understanding of lung biology, is that lung HSPCs, like spleen HSPCs, are less active in terms of cell division and the production of more differentiated and specialized hematopoietic cells than their BM counterparts. 31These results from in vitro experiments would suggest that lung HSPCs function as a reserve pool that could be mobilized in the setting of hematopoietic stress.While physiologically most lung HSPCs might be silenced, a case of pulmonary hematopoesis after sex-mismatched allogeneic stem cell transplantation (SCT) suggests that indeed engraftment into the lung occurs and can lead to active hematopoeisis years after SCT. 32Further studies are needed to de ne physiological and pathological stimuli that trigger hematopoiesis in the lung.
Our xenotransplantation experiments indicated that lung HSPCs performed similarly to medullary HSPCs, when given the challenge of engraftment after sublethal irradiation.A wide range of engraftment levels and analysis time points have been used for measuring human HSC activity in vivo and there is no standard in the eld. 16Thus, despite low graft expansion in our model (NSG-SGM3 mice, sub-lethal irradiation, intravenous HSPCs), our data are consistent with previous outcomes and demonstrate selfrenewal capacity of pulmonary HSPCs in vivo.Additionally, we included donors of any age and sex (Table S1), which might have contributed to larger variations in our results.
Our transcriptomics analysis revealed a distinct molecular program in lung HSPCs that was further distinguishing compared to medullary HSPCs.We found clear megakaryocyte biasing of lung vs. medullary HSPCs indicating that perhaps the lung is a source of these biased progenitors.This is an intriguing nding given (1) the role of the lung in platelet biogenesis and (2) the presence of tissue resident immune-like megakaryocytes (of unclear ontogeny) in the lung. 7We further found an erythroid bias of lung HSPCs, akin to that reported in peripheral blood and spleen HSPCs. 31Erythroid bias therefore is a shared feature of steady-state extramedullary HSPCs, perhaps determined by distinct access to environmental oxygen versus the relatively hypoxic environment of the BM. 33r spatial transcriptomic studies were essential in con rming the presence of HSPCs in the lung and de ning their precise locations and niche.Given the prevailing dogma that hematopoietic precursors widely circulate [34][35][36] , it was important to rule out that blood contamination was producing our results, although our results in mice indicated an extravascular location. 7We found a few intravascular HSPCs, con rming previous studies, but the vast majority were extravascular and predominately in vascular-rich zones of the lung alveoli.This anatomic location in the lung is similar to the location of HSPCs in the BM, which are closely positioned next to the vascular sinusoids. 37,38In the lung, this positioning could be important for seeding of the lung with circulating HSCs and potentially for exiting the lung during hematopoietic stress.In this niche, it was notable that lung broblasts were in close proximity.The lung mesenchyme is well known to be a critical niche supporting epithelial cell development and repair and similar mechanisms could be operable in uencing lung HSPCs. 39,40Also, we identi ed a developmental trajectory between lung HSPCs and lung stromal cells.Subpopulations of lung broblasts are known to be CD34 + and previous lineage-tracing experiments in pulmonary brosis have shown a hematopoietic contribution to broblasts. 41,42Future studies are needed in this area to understand how lung HSPCs could be involved in brotic lung diseases.
We have not addressed the ontogeny of lung HSPCs-something not possible given the restraints of our human tissue study.There is irrefutable evidence that HSCs commonly enter the circulation and microcirculatory beds, and given the functional and molecular similarities of lung HSPCs to other extramedullary HSPCs, this is perhaps the source of the tissue resident HSPCs in the lung. 35,43There is precedence, however, for tissue residency to be endowed during development, such as with yolk-sac derived macrophages. 44Future studies will be needed to answer this question in mice, including the possibility that hemogenic endothelium in the fetal lung could be the source. 45r ndings reframe our understanding of the HSPC pool and its molecular diversity and should enable future studies that could potentially lead to therapeutic advances, such as for life-saving HSC transplantation for BM malignancies and failure.In the modern era, transplantation is mainly accomplished using mobilized HSCs obtained from the peripheral blood.We propose that these mobilized cells are sourced from diverse tissues, including the lung, and that the composition of this pool may be functionally heterogeneous, which could have important implications for treatment responses and complications.In this regard, our ndings may also help to understand the mechanisms of leukemogenesis with the possibility that lung HSPCs are direct targets of environmental carcinogens. 46rther, our ndings add to our expanding understanding of rare cell types in the lung and their potential functions. 47

Declarations Figures
The Stacked bars represent mean proportion ± SD, ANOVA followed by Sidak's multiple comparison test, **p<0.01,*p<0.05.For comparisons not indicated, no statistically signi cant differences were observed.